The acidic polysaccharides known as glycosaminoglycans (GAGs), including heparin/heparan sulfate (HS), chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronic add, present in connective tissue and the cell membrane, aggregate around a core protein with covalent bond to form proteoglycans (PGs). PGs form extracellular matrices (ECMs) with cell-adhesive proteins such as fibronectin, vitronectin, laminin, collagen and thrombospondin, and are widely distributed for supporting cell survival and physiological functions of cells. In particular, heparan sulfate proteoglycans (HSPGs) are present in almost all animal tissues, and perform a crucial role in the processes of cell adhesion, morphogenesis and maintenance of function.
It has become apparent that the heparin/HS found in PGs interact with various cell growth factors to control cell differentiation and proliferation. For example, fibroblast growth factors (FGFS) which have a high affinity to heparin/HS constitutes the FGF family (FGF1–FGF10 have been reported to date), and act specifically with respect to vascular endothelial cells, Kaposi's sarcoma cells and epidermal keratinized cells depending on their type. The activities of FGFs are believed to be triggered by binding specifically to FGF receptors (FGFRS) on the cell surfaces. Heparin/HS, which is present transmembranously, holds and stores unstable FGF molecules in a stable state in the vicinity of the cell, and support binding of the FGFs to the FGFRs as needed while protecting the FGFs from proteases and oxidative decomposition. The binding of FGFs to FGFRs causes proliferation signals to be generated, thus accelerating cell proliferation. This action mechanism is suggested by a large number of studies indicating that FGFs and FGFRs cannot bind unless heparin/HS is present (e.g., M. Ishihara, Glycobiology, 4, 817–824, 1994).
On the other hand, chondroitin sulfate exists in abundance in the massive hyaluronic acid-rich PG backbones found in cartilage tissue, and is highly involved with the control of osteogenesis. Thus, GAGs are distributed and structured in various tissues according to their function, controlling the growth of specific cells.
The present inventors performed basic research into GAGs including heparin/HS which have such diverse functions and especially into their application to medicine, in the process of which they synthesized glycosaminoglycan-functionalized polymers formed by binding the backbones of GAGs to vinyl polymer main chains, and filed a patent application to cover their use as cell culture media and anti-tumor agents (WO 00/59967). This type of functionalization enables the activity of cell growth possessed by GAGs to be efficiently enhanced.
On the other hand, cell-adhesive proteins, collagen among them, which are the main ingredients forming extracellular matrices together with PGs, are commonly used in cell culture substrates and artificial organs for their cell-adhesive properties. For example, various cell types including fibroblasts, endothelial cells and neutrophils have been demonstrated to adhere to matrices consisting only of collagen, where they can grow or migrate. Additionally, this type of adhesion has been shown to depend on the number of cell surface receptors such as members of the integrin family (Myles, J. L. et al., J. Biomater Sci. Polymer Edn., 11:69–86, 2000).
In the field of tissue remodeling which has lately been the focus of increased interest, various attempts have been made to control differentiation/proliferation of various types of cells ex vivo. In general, differentiation gives preference to the expression of functions specific to the cell while inhibiting cell proliferation, and proliferation (dedifferentiation) gives preference to multiplication over function. In the process of tissue regeneration, it is important to maintain a balance between proliferation (dedifferentiation) and the expression of specific functions due to differentiation. Generally, collagen is considered to tend to induce dedifferentiation during in vitro cell cultivation.